Combined ultrahigh-resolution optical tweezers and single-molecule fluorescence

描述（由申请人提供）：单分子技术已经发展成为研究许多基本生物过程中涉及的分子机器的有力工具。诸如荧光定位、F“rster共振能量转移（FRET）和光学镊子的技术已经有助于破译分子马达的机制，所述分子马达诸如肌球蛋白和驱动蛋白、DNA和RNA聚合酶以及解旋酶，仅举几个例子。最近，超高分辨率光镊的发展首次使直接观察DNA的1个碱基对（3.4 <$）尺度上的分子运动成为可能。尽管有这些进展，单分子技术仍有重要的局限性。虽然分子机器中涉及的构象变化本质上是三维的，但这种技术通常将所有运动投射到单个轴上，因此无法捕获分子运动的全部复杂性。此外，这些技术在很大程度上被限制到简单的系统，涉及很少的组件孤立地检查，而在细胞的情况下，分子机器由高度协调的多组分蛋白质组装。 为了解决这些局限性，我们建议1）开发新一代的单分子仪器，结合多色荧光检测和超高分辨率光镊。虽然仪器合并荧光和光学陷阱已经开发出来，实现碱基对分辨率仍然是一个巨大的挑战，将需要一个新的方法。然而，这些能力对于理解参与DNA代谢的分子复合体（转录、复制、重组和修复）是必不可少的，这些分子复合体具有重大的生物医学意义。我们提出的混合工具将有能力同时测量多个可观的，如内部蛋白质动力学的FRET或组装动力学的蛋白质复合物的荧光定位，结合检测电机位移在碱基对分辨率的光镊。作为该技术的示范，我们将2）监测易位和双链体解旋的E。coliRep解旋酶的碱基对分辨率，同时通过FRET和寡聚体状态的构象变化，通过荧光检测。这项拟议中的工作涉及超高分辨率光镊专家的合作（Y。Chemla，PI）和单分子荧光（T.哈，合作PI）在伊利诺伊大学香槟分校。 公共卫生相关性声明：我们正在计划开发一种仪器，它将联合收割机结合两种强大的尖端技术：单分子荧光和超高分辨率光学捕获。我们的目标是研究蛋白质和蛋白质复合物的动力学参与DNA复制，转录，重组和修复在<$ngstrom水平的分辨率。这项技术有可能揭示这些分子机器的详细机制，这是一个具有巨大医学意义的问题，因为它们的活性缺陷与许多人类疾病，特别是癌症有关。

DESCRIPTION (provided by applicant): Single molecule techniques have developed into a powerful tool to study the molecular machines involved in many fundamental biological processes. Techniques such as fluorescence localization, F"rster resonance energy transfer (FRET), and optical tweezers have been instrumental in deciphering the mechanism of molecular motors such as myosin and kinesin, DNA and RNA polymerases, and helicases, to name just a few examples. Recently, the development of ultrahigh-resolution optical tweezers has made possible, for the first time, the direct observation of molecular motion on the scale of 1 basepair of DNA (3.4¿). Despite such advances, single molecule techniques have had important limitations. Although the conformation changes involved in molecular machines are inherently three-dimensional, such techniques typically project all motion onto a single axis and thus cannot capture the full complexity of molecular motion. Furthermore, these techniques have largely been limited to simple systems involving very few components examined in isolation, whereas, in the cellular context, molecular machines consist of highly coordinated multi-component protein assemblies. To address these limitations, we propose to 1) develop the new generation of single molecule instrumentation combining multi-color fluorescence detection and ultrahigh-resolution optical tweezers. Although instruments merging fluorescence and optical traps have been developed previously, achieving basepair resolution remains a grand challenge that will require a new approach. These capabilities nevertheless will be essential to understand the molecular complexes involved in DNA metabolism-transcription, replication, recombination, and repair-that have great biomedical significance. The hybrid instrument we propose will have the ability to measure multiple observables simultaneously, such as internal protein dynamics by FRET or the assembly kinetics of protein complexes by fluorescence localization, combined with detection of motor displacement at basepair resolution by optical tweezers. As a demonstration of this technique, we will 2) monitor translocation and duplex unwinding by E. coli Rep helicase at basepair resolution, simultaneously with conformational changes by FRET and oligomeric state by fluorescence detection. This proposed work involves the collaboration of experts in ultrahigh-resolution optical tweezers (Y. Chemla, PI) and single-molecule fluorescence (T. Ha, co-PI) at the University of Illinois, Urbana-Champaign. Public Health Relevance Statement: We are proposing to develop an instrument that will combine two powerful cutting-edge technologies: single-molecule fluorescence and ultrahigh-resolution optical trapping. Our goal is to study the dynamics of proteins and protein complexes involved in DNA replication, transcription, recombination, and repair at ¿ngstrom level resolution. This proposed technique has the potential to reveal the detailed mechanism of these molecular machines, a problem of great medical interest as defects in their activity have been implicated in a number of human diseases, specifically cancer.